Estudo prospectivo para identificar a presença do papilomavírus humano em gestantes adolescentes por meio da captura híbrida II e correlacionar com colpocitologia oncótica convencional, em base líquida e colposcopia. O grupo constituído por 60 gestantes entre 12 a 18 anos e idade gestacional media de 23 semanas. A captura híbrida II foi positiva em 51,7%. A colpocitocologia oncótica convencional : normal em 90% e anormal em 10%. Os achados citológicos anormais: lesão intraepitelial escamosa de baixo grau em 8,3% e carcinoma invasor em 1,7%. A colpocitologia em base líquida: normal em 90% e anormal em 10%. Os achados citológicos anormais: lesão intraepitelial escamosa de baixo grau em 8,3% e lesão intraepitelial escamosa de alto grau em 1,7%. Os achados colposcópicos normais foram o epitélio escamoso normal em 20%, epitélio glandular em 18,3% e a zona de transformação normal em 40%. A zona de transformação anormal presente em 21,7% / Study delineated to identify human papillomavirus by hybrid capture II in pregnant teenagers and to correlate with conventional, liquid-based Pap test and colposcopic findings. The study group was constituted by 60 pregnant women aged between 12 and 18 years old; mean gestational age was 23 weeks. They were submitted to anamnese, Pap smear, hybrid capture technique and colposcopy. Hybrid capture II of human papillomavirus was positive in 51,7% of the cases. Conventional Pap test was normal in 90% and abnormal in 10% of the cases. Abnormal results: low-grade squamous intraepithelial lesion in 8,3% and Invasor carcinoma in 1,7%. Based-liquid Pap test was normal in 90% and abnormal in 10% of the cases. Abnormal results: 8,3% of low-grade squamous intraepithelial lesion and 1,7% of high-grade squamous intraepithelial lesion. Normal results: 20% of normal squamous epithelium, 18,3%of columnar epithelium and 40% of normal transformation zone. Abnormal transformation zone was seen in 21,7% of the cases

La membrane bactérienne a fait l'objet de nombreuses études de localisation de protéines et de phospholipides. Par fusion d’une protéine fluorescente (GFP) aux gènes d’intérêt, il est alors possible d‘observer la localisation des protéines associées par microscopie. La plupart de ces observations ont été réalisées à l’aide de microscopes dits à épifluorescence. Afin d’obtenir une qualité d’image suffisante, il était nécessaire de surexprimer la protéine observée, insérée à un locus ectopique non naturel. Ce travail de thèse a permis d’utiliser une nouvelle technologie acquise dans notre laboratoire, le microscope à fluorescence par réflexion totale interne (TIRFM), plus puissant que le microscope à épifluorescence utilisé précédemment. Cette technologie a permis une caractérisation plus détaillée de la localisation de protéines d’intérêt, placées sous contrôle de leur promoteur naturel. Il a également été possible de caractériser la dynamique des foci observés. Nous avons concentré notre étude sur 3 protéines: (i) SecA pour l’étude de la translocation des protéines du cytoplasme vers la membrane, (ii) YidC pour l’insertion des protéines dans la membrane, (iii) PgsA pour la synthèse des phospholipids. Les foci se déplacent dynamiquement et s’associent de manière transitoire dans la membrane. L’observation sur la durée de ces foci, et l’analyse de leur intensité moyenne au cours des observations, montre que SecA se déplace sur l’ensemble de la membrane de manière uniforme. L’analyse du déplacement des foci montre une relation quadratique entre la distance moyenne parcourue par les foci en fonction du temps. Ce résultat est en accord avec l’hypothèse d’un mouvement brownien des foci. Les foci sont observés dans les différentes phases de croissance des cellules, et le nombre de foci présents dans une cellule de la longueur de celle-ci. SecA-GFP a été testés dans un certain nombre de contextes génétiques. La localisation a été perturbée lors de la déplétion de pgsA. Cependant, comme PgsA est une protéine essentielle, il ne peut être exclu que ce changement de localisation apparaît des cellules qui sont mortes ou mourantes. Dans une souche mutante ΔclsA, on n’observe aucune différence dans la localisation de SecA en phase exponentielle, mais on aperçoit une relocalisation aux poles en phase stationnaire de croissance. La voie Tat est responsable du transport des protéines devant être exportées dans un état structuré, par exemple dans le cas de l’incorporation d’un co-facteur. À ce jour, la régulation du système Tat est peu connues, de même que les interactions entre les différentes sous-unités du système Tat et d'autres protéines dans le cytoplasme, dans la membrane ou dans la paroi cellulaire. Des fusions de les gènes de la voie Tat ont été co-exprimées deux à deux dans des cellules de levure, et leur capacité à interagir in vivo a été testée par la méthode dite du double hybride chez la levure. Nous avons généré un réseau d’interaction autour des cinq composants de système Tat. Pour déterminer les implications fonctionnelles des composants du réseau, nous avons travaillé en collaboration avec le laboratoire de Jan-Maarten van Dijl. Nous avons utilisé une collection de souches mutantes pour lesquels certains composants individuels du réseau ont été retires. Trois a été observe d’etre nécessaires pour la sécrétion Tat-dépendante. Nous avons étudié la localisation des fusions GFP avec ces proteins. On a observé une localisation double de HemAT selon l’état physiologique de la cellule. En phase exponentielle, les cellules de B. subtilis sont généralement présentes sous forme de chaînes dans lesquelles le septum de division a déjà été formé, mais la séparation cellulaire n'a pas encore eu lieu. Une fusion de la GFP à CsbC apparaît de façon homogène dans la membrane. / In the years since the cloning of GFP, the field of bacterial cell biology has characterized a variety of specific protein localization patterns in the bacterial membrane. The vast majority of early subcellular localization studies made use of inducible GFP fusions, which generally required the presence of high concentrations of inducer, and can therefore be considered to be overexpressed. An outstanding question remains over the organization of natively expressed proteins in the membrane. Here, we have investigated the localization of functional GFP fusions to proteins catalyzing important membrane processes; the secretion motor protein SecA, the membrane insertase YidC1, and the essential phospholipid synthase PgsA using total internal reflection fluorescence microscopy (TIRFM). This allowed natively expressed proteins to be localized with temporal resolution that can capture their dynamics. We characterized dynamic complexes dispersed throughout the membrane displaying diffusive movement with no preferred trajectories. Further characterization focused upon identifying conditions in which the localization pattern was disturbed. A polar mislocalization was identified in a cardiolipin mutant strain. The yeast two-hybrid (Y2H) approach is a robust approach to detect binary interactions on a proteome-scale. We performed genome-wide Y2H screens as well as targeted Y2H analyses for specific interactions involving components of the Sec and Tat secretion machineries of B. subtilis, revealing an intricate protein-protein interaction network involving 71 proteins. Furthermore, three proteins identified in the Tat network, WprA, CsbC and HemAT, were shown to be important for effective protein secretion via the B. subtilis Tat system, indicating that our yeast two hybrid assays reveal biologically significant interactions involving membrane proteins. The studies provide a novel proteomic view on the interaction network of the secretion systems of B. subtilis.

Made available in DSpace on 2016-01-26T12:51:42Z (GMT). No. of bitstreams: 1 arielifernandagavioligomes_dissert.pdf: 1557143 bytes, checksum: 898e0714fcf73f6bc8ba53f5719efc71 (MD5) Previous issue date: 2011-12-15 / Conselho Nacional de Desenvolvimento Científico e Tecnológico / Introduction: The Yellow Fever is characterized by severe hepatitis, renal failure, hemorrhage, and rapid terminal events that lead to shock and death. This disease is caused by the infection with the Yellow Fever Virus (YFV), considered the prototype of the Flavivirus genus. Its mechanism of replication is not well known but includes interactions of viral RNA with cellular and viral proteins. The nonstructural protein 5 (NS5) is the largest and most conserved protein of the Flavivirus genus; it encodes RNA-dependent RNA polymerase (RdRp) domains, besides possessing many important functions during viral replication, such as genic regulation of host cells. The protein hSlu7 is a homologous human protein, which was isolated interacting with the U5 that is involved in the second the step of alternative splicing. The hSlu7 is a predominantly nuclear protein and participates at the alternative splicing, influencing the correct choice of the alternative AGs of exon 3' so that the spliceossome is able to bind and start alternative splicing. Objective: To characterize the interaction of the hSlu7 protein with the YFV-NS5 protein and its cellular localization during viral infection. Material and Method: We confirmed the interaction of various NS5 with human proteins by two-hybrid assays. Deletion mutants were constructed and co-transformed with hSlu7 in yeast to determine the minimal domain of NS5 required for interaction. The cellular localization of the hSlu7 fused with GFP during the response of vaccine strain 17D of YFV in cells Vero E6, marked with anti-NS4AB and anti-NS5 for detection of the infection was also tested. Results: hSlu7 interacts with initial and final portions of the RdRp and the cytoplasmic sublocalization of hSlu7 occurs in the cells infected with YFV. Conclusions: Our results suggest that hSlu7 interacts with the YFV by two-hybrid system and the cellular sublocalization occurs due to the presence of viral infection. Further studies using RNA interference should be addressed to confirm the cellular function of hSlu7 , and to evaluate which alterations that infected and uninfected cells will suffer with low levels of hSlu7. / Introdução: A Febre Amarela é uma doença decorrente da infecção pelo Vírus da Febre Amarela (YFV) que é um protótipo do gênero Flavivirus que provoca uma severa hepatite, falência renal, hemorragia, e eventos que rapidamente levam ao choque e morte do indivíduo. Os mecanismos de replicação genômico do YFV não são bem conhecidos. A proteína não estrutural 5 (NS5) é a maior proteína e a mais conservada dos Flavivirus, ela codifica a RNA polimerase dependente de RNA (RdRp). A hSlu7 é uma proteína celular, predominantemente nuclear, e foi isolada interagindo com a U5 no segundo passo do splicing alternativo. A hSlu7 auxilia na correta seleção dos AGs alternativos do exon 3 para a realização da reação de splicing. Objetivo: Caracterizar a interação de hSlu7 com a proteína NS5 de YFV, quanto a sua localização celular durante a infecção. Material e Método: Pelo sistema duplo-híbrido em leveduras utilizando plasmid-linkage avaliamos a interação de hSlu7 com deleções mutantes da RdRp de YFV, e a localização celular de GFP-hSlu7 durante a replicação da cepa vacinal 17D de YFV em cultura de células Vero E6, marcadas com anticorpos NS4AB e NS5 para detecção da infecção. Resultados: A hSlu7 interage com as porções inicial e final da RdRp, e a sublocalização citoplasmática de hSlu7, ocorre nas células infectadas com YFV. Conclusão: Nossos resultados sugerem que a hSlu7 possui uma sublocalização celular durante a replicação do YFV, além de interagir com a RdRp viral. Ainda será necessária a confirmação da função celular de hSlu7 utilizando RNA de interferência para avaliar quais as alterações que a célula não infectada e infectada sofrerá diante dos níveis baixos de hSlu7.

Estudo prospectivo para identificar a presença do papilomavírus humano em gestantes adolescentes por meio da captura híbrida II e correlacionar com colpocitologia oncótica convencional, em base líquida e colposcopia. O grupo constituído por 60 gestantes entre 12 a 18 anos e idade gestacional media de 23 semanas. A captura híbrida II foi positiva em 51,7%. A colpocitocologia oncótica convencional : normal em 90% e anormal em 10%. Os achados citológicos anormais: lesão intraepitelial escamosa de baixo grau em 8,3% e carcinoma invasor em 1,7%. A colpocitologia em base líquida: normal em 90% e anormal em 10%. Os achados citológicos anormais: lesão intraepitelial escamosa de baixo grau em 8,3% e lesão intraepitelial escamosa de alto grau em 1,7%. Os achados colposcópicos normais foram o epitélio escamoso normal em 20%, epitélio glandular em 18,3% e a zona de transformação normal em 40%. A zona de transformação anormal presente em 21,7% / Study delineated to identify human papillomavirus by hybrid capture II in pregnant teenagers and to correlate with conventional, liquid-based Pap test and colposcopic findings. The study group was constituted by 60 pregnant women aged between 12 and 18 years old; mean gestational age was 23 weeks. They were submitted to anamnese, Pap smear, hybrid capture technique and colposcopy. Hybrid capture II of human papillomavirus was positive in 51,7% of the cases. Conventional Pap test was normal in 90% and abnormal in 10% of the cases. Abnormal results: low-grade squamous intraepithelial lesion in 8,3% and Invasor carcinoma in 1,7%. Based-liquid Pap test was normal in 90% and abnormal in 10% of the cases. Abnormal results: 8,3% of low-grade squamous intraepithelial lesion and 1,7% of high-grade squamous intraepithelial lesion. Normal results: 20% of normal squamous epithelium, 18,3%of columnar epithelium and 40% of normal transformation zone. Abnormal transformation zone was seen in 21,7% of the cases

Dechamps Christophe (2008). Etude fonctionnelle dun oncogène humain impliqué dans le Sarcome dEwing, oncTre210, homologue à deux gènes levuriens, MSB3 et MSB4 (thèse de doctorat). Gembloux, Faculté Universitaire des Sciences Agronomiques, 173 p., 12 tabl., 40 fig. Résumé : Le produit de l'oncogène humain oncTre210 est apparenté, par sa structure primaire, aux protéines Ypt/Rab GAP (GTPase Activating Proteins spécifiques des Ypt/Rab GTPases). En effet, sa région N-terminale, qui est fortement homologue aux deux GAP de Saccharomyces cerevisiae Msb3p et Msb4p, renferme le domaine catalytique TBC, hautement conservé, des Ypt/Rab GAP. Les protéines Msb3p et Msb4p de Saccharomyces cerevisiae font partie de la famille des GTPase activating protein (GAP) spécifique aux Ypt/Rab GTPase. Elles sont primordiales au trafic vésiculaire et sont impliquées dans la régulation de l'exocytose et dans l'organisation du cytosquelette d'actine. Mais, leurs rôles biologiques exacts nont jamais été déterminés. La délétion simultanée des 2 gènes MSB3 et MSB4 dans la levure S. cerevisiae induit une inhibition de croissance de la levure sur un milieu de culture contenant du DMSO et/ou de la caféine, perturbe lorganisation du cytosquelette dactine, produit une anomalie de bourgeonnement dans la levure diploïde et affecte la ségrégation des noyaux. Pour trouver des composants qui interagissent génétiquement avec les produits des gènes MSB3 et MSB4, nous avons criblé une banque génomique pour des suppresseurs homologues extragéniques multicopies restaurant la croissance du double mutant levurien msb3 msb4 en présence de DMSO et/ou de caféine. Sept gènes ont ainsi été identifiés après une série de vérifications. Ces 7 suppresseurs peuvent être classés pour la fonction biologique de leurs produits en plusieurs classes : transport vésiculaire, cycle cellulaire, chaperon moléculaire, protéasome et ARN ribosomial. Ces résultats nous permettent d'identifier les voies physiologiques où les deux protéines Msb3p et Msb4p seraient impliquées. Le produit de l'oncogène oncTre210 est impliqué dans différents cancers humains dont le sarcome d'Ewing. Pour létude des partenaires interagissant avec lune ou lautre des deux parties de la protéine de fusion oncTre210p, nous avons utilisé le système double-hybride en levure en utilisant différentes banques d'expression. Un grand nombre de partenaires protéiques a été isolé comme interagissant avec l'oncoprotéine. Deux protéines impliquées dans l'organisation et la structure du cytosquelette ont été choisies parmi les partenaires de l'oncoprotéine pour être étudiées. L'interaction de ces deux protéines avec la partie GAP de oncTre210p a été confirmée par les techniques de GST pulldown, de co-immunoprécipitation et de co-localisation. Ces protéines identifiées comme interagissant avec la partie GAP sont la chaîne légère régulatrice de la myosine II (Myl2) et LOC91256, protéine contenant des motifs ankyrine. A partir de ces observations un nouveau rôle de l'oncoprotéine oncTre210p a été suggéré. L'ensemble de nos résultats ainsi que des données expérimentales acquises par d'autres équipes internationales nous a permis de proposer un modèle d'action pour l'oncoprotéine oncTre210p. Dechamps Christophe (2008). Functional study of an oncogene implicated in Ewing's sarcoma, oncTre210, a human homologue of two yeast genes, MSB3 and MSB4 (Thesis in French). Gembloux, Belgium, Gembloux Agricultural University, 173 p., 12 tabl., 40 fig. Summary : The oncTre210 oncogene product is structurally related to the Ypt/Rab GTPase-Activating Proteins (Ypt/Rab GAPs). Particularly, the N-terminal region of the oncoprotein shares with the yeast proteins Msb3p and Msb4p the highly conserved TBC domain, forming the catalytically active domain of Ypt/Rab GAPs. The Msb3p and Msb4p proteins of Saccharomyces cerevisiae are members of the Ypt/Rab-specific GTPase activating protein (GAP) family. They are important to vesicular trafficking and involved in the regulation of exocytosis and in the organization of the actin cytoskeleton, but their exact biological roles have yet to be determined. The msb3 msb4 yeast double mutation causes growth inhibition in the presence of DMSO and/or caffeine, affects the organization of the actin cytoskeleton, produces a random budding pattern in diploid cells, and affects segregation of the nucleus. To find cell components that interact genetically with the products of the MSB3 and MSB4 genes, we screened a genomic library for multicopy suppressor genes restoring normal growth of the double mutant in the presence of DMSO and caffeine. Six genes were identified, and the extent to which each gene corrects specific growth defects of the msb3 msb4 mutant is described. The encoded suppressors were classified on the basis of functional features into five groups: vesicular transport, cell division, molecular chaperon, proteasome and ribosomal RNA. These results allow us to identify the physiologic ways where the Msb3p and Msb4p proteins are implicated. The product of the oncTre210 oncogene is involved in various human cancers, including Ewings sarcoma. In order to identify proteins interacting with the two parts of this protein, we performed yeast two-hybrid screening of various libraries. A large number of proteins was identified to be partners of the oncogene product. Two components of the cytoskeleton were chosen to be studied, whose interaction with the GAP region was confirmed by GST-pulldown, co-immunoprecipitation, and colocalisation experiments. The proteins found to interact with the GAP region are the light regulatory chain of myosin II (Myl2) and LOC91256, a protein containing ankyrin repeats. From these observations a new role for the oncTre210p oncoprotein in cytokinesis was suggested. Our results and data from other international teams allow us to propose a model for the action of the oncTre210 oncogene product.

Ubiquitylation is an indispensable post-translational modification system in eukaryotic cells that leads to the covalent attachment of a small ubiquitin (Ub) protein onto a target. The traditional and best-characterized role for ubiquitylation is a fundamental regulatory mechanism whereby target proteins are tagged with a characteristic Lys48-linked Ub chain that signals for their elimination through proteasomal degradation. Challenging this conventional wisdom is the finding that some ubiquitylated proteins are modified by Ub chains linked through Lys63, providing a molecular signal that is thought to be structurally and functionally distinct from Lys48-linked Ub chains. Of further interest and significance is that the Lys63-linked Ub chains are apparently synthesized through a novel biochemical mechanism employing a unique complex formed between a true Ub conjugating enzyme (E2), Ubc13, and an E2-variant (Uev), Mms2 (or Uev1A). The goal of this thesis was to employ structural and functional approaches in order to better characterize the Ubc13-Mms2 Lys63-linked Ub conjugation complex. <p>Error-free DNA damage tolerance (DDT) in the budding yeast is dependent on Lys63-linked Ub chains synthesized by Ubc13-Mms2 and thus provided the opportunity to experimentally test the function of the human UBC13 and MMS2 genes in a simple model organism. Human UBC13 and MMS2 were each shown to function in place of their yeast counterparts and in accordance, human Ubc13 was shown to physically interact with yeast Mms2, and vice versa. Two human MMS2 homologs were also tested and it was determined that UEV1A but not UEV1B can function in place of mms2 in yeast DDT. Physical interactions were observed between Ubc13 and Uev1A, but not between Ubc13 and Uev1B, suggesting that Ubc13-Uev complex formation is required for function. <p>In collaboration with a research group at the University of Alberta, crystal structure and NMR data were used to develop a mechanistic model for the conjugation of Lys63-linked Ub chains by the Ubc13-Mms2 heterodimer, whereby the special orientation of two Ub molecules facilitates a specific Ub-Ub linkage via Lys63. In order to help support the in vitro model and to determine how the Ubc13-Mms2 structure relates to biological function, I used a structure-based approach to direct the creation of point mutations within four key regions of the Ubc13-Mms2 heterodimer; the Ubc13 active-site, the Ubc13-E3 (Ub ligating enzyme) interface, the Mms2-Ub interface, and the Ubc13-Mms2 interface. <p>Underscoring the importance of the Ub conjugation by Ubc13-Mms2, a Ubc13-C87S active-site mutation was created that could bind to Mms2 but was unable to function in DDT. Regarding the Ubc13-E3 interface, a single Ubc13-M64A point mutation had a potent effect on disrupting Ubc13 function in DDT, as well as its physical interaction with Rad5, TRAF6, and CHFR. The results suggest that different RING finger E3s use the same Ubc13 surface to sequester the Ub conjugation activity of Ubc13-Mms2. Two human Mms2 mutations at Ser32 and Ile62, which are contained within the Mms2-Ub interface, were found to reduce the ability of Mms2 to bind Ub. When the corresponding yeast mutations are combined, a synergistic loss in DDT function is observed. The relative orientation of Ser32 and Ile62 suggests that the Mms2 and Tsg101 Uev families use different Uev surfaces to physically interact with Ub. A 200 ìM dissociation constant for the wild-type Mms2-Ub interaction was also determined. The systematic mutagenesis and testing of 14 Ubc13-Mms2 interface residues led to mutants with partial or complete disruption of binding and function. Using this data, a model involving the insertion of a specific Mms2-Phe residue into a unique Ubc13 hydrophobic pocket was created to explain the specificity of Mms2 for Ubc13, and not other E2s. In addition, the dissociation constant for the wild-type Ubc13-Mms2 heterodimer was determined to be approximately 50 nM. <p>The structural and functional studies strongly support the notion that Ubc13-Mms2 complex has the unique ability to conjugate Lys63-linked Ub chains. However, several reported instances of Lys63-linked Ub chains in vivo have not yet been attributed to Ubc13 or Mms2. To address the disparity I was able to demonstrate and map a physical interaction between Mms2 and Rsp5, an E3 implicated in Lys63-linked Ub conjugation. Surprisingly, it was found that MMS2 is not responsible for the RSP5-dependent Lys63-linked Ub conjugation of a plasma membrane protein. A possible explanation for the apparent paradox is presented.

Ubiquitylation is an indispensable post-translational modification system in eukaryotic cells that leads to the covalent attachment of a small ubiquitin (Ub) protein onto a target. The traditional and best-characterized role for ubiquitylation is a fundamental regulatory mechanism whereby target proteins are tagged with a characteristic Lys48-linked Ub chain that signals for their elimination through proteasomal degradation. Challenging this conventional wisdom is the finding that some ubiquitylated proteins are modified by Ub chains linked through Lys63, providing a molecular signal that is thought to be structurally and functionally distinct from Lys48-linked Ub chains. Of further interest and significance is that the Lys63-linked Ub chains are apparently synthesized through a novel biochemical mechanism employing a unique complex formed between a true Ub conjugating enzyme (E2), Ubc13, and an E2-variant (Uev), Mms2 (or Uev1A). The goal of this thesis was to employ structural and functional approaches in order to better characterize the Ubc13-Mms2 Lys63-linked Ub conjugation complex. <p>Error-free DNA damage tolerance (DDT) in the budding yeast is dependent on Lys63-linked Ub chains synthesized by Ubc13-Mms2 and thus provided the opportunity to experimentally test the function of the human UBC13 and MMS2 genes in a simple model organism. Human UBC13 and MMS2 were each shown to function in place of their yeast counterparts and in accordance, human Ubc13 was shown to physically interact with yeast Mms2, and vice versa. Two human MMS2 homologs were also tested and it was determined that UEV1A but not UEV1B can function in place of mms2 in yeast DDT. Physical interactions were observed between Ubc13 and Uev1A, but not between Ubc13 and Uev1B, suggesting that Ubc13-Uev complex formation is required for function. <p>In collaboration with a research group at the University of Alberta, crystal structure and NMR data were used to develop a mechanistic model for the conjugation of Lys63-linked Ub chains by the Ubc13-Mms2 heterodimer, whereby the special orientation of two Ub molecules facilitates a specific Ub-Ub linkage via Lys63. In order to help support the in vitro model and to determine how the Ubc13-Mms2 structure relates to biological function, I used a structure-based approach to direct the creation of point mutations within four key regions of the Ubc13-Mms2 heterodimer; the Ubc13 active-site, the Ubc13-E3 (Ub ligating enzyme) interface, the Mms2-Ub interface, and the Ubc13-Mms2 interface. <p>Underscoring the importance of the Ub conjugation by Ubc13-Mms2, a Ubc13-C87S active-site mutation was created that could bind to Mms2 but was unable to function in DDT. Regarding the Ubc13-E3 interface, a single Ubc13-M64A point mutation had a potent effect on disrupting Ubc13 function in DDT, as well as its physical interaction with Rad5, TRAF6, and CHFR. The results suggest that different RING finger E3s use the same Ubc13 surface to sequester the Ub conjugation activity of Ubc13-Mms2. Two human Mms2 mutations at Ser32 and Ile62, which are contained within the Mms2-Ub interface, were found to reduce the ability of Mms2 to bind Ub. When the corresponding yeast mutations are combined, a synergistic loss in DDT function is observed. The relative orientation of Ser32 and Ile62 suggests that the Mms2 and Tsg101 Uev families use different Uev surfaces to physically interact with Ub. A 200 ìM dissociation constant for the wild-type Mms2-Ub interaction was also determined. The systematic mutagenesis and testing of 14 Ubc13-Mms2 interface residues led to mutants with partial or complete disruption of binding and function. Using this data, a model involving the insertion of a specific Mms2-Phe residue into a unique Ubc13 hydrophobic pocket was created to explain the specificity of Mms2 for Ubc13, and not other E2s. In addition, the dissociation constant for the wild-type Ubc13-Mms2 heterodimer was determined to be approximately 50 nM. <p>The structural and functional studies strongly support the notion that Ubc13-Mms2 complex has the unique ability to conjugate Lys63-linked Ub chains. However, several reported instances of Lys63-linked Ub chains in vivo have not yet been attributed to Ubc13 or Mms2. To address the disparity I was able to demonstrate and map a physical interaction between Mms2 and Rsp5, an E3 implicated in Lys63-linked Ub conjugation. Surprisingly, it was found that MMS2 is not responsible for the RSP5-dependent Lys63-linked Ub conjugation of a plasma membrane protein. A possible explanation for the apparent paradox is presented.

Nuclear receptors are ligand-activated transcription factors that play significant roles in various biological processes within the body, such as cell development, hormone metabolism, reproduction, and cardiac function. As transcription factors, nuclear receptors are involved in many diseases, such as diabetes, cancer, and arthritis, resulting in approximately 10-15% of the pharmaceutical drugs presently on the market being targeted toward nuclear receptors. Structurally, nuclear receptors consist of a DNA-binding domain (DBD), responsible for binding specific sequences of DNA called response elements, fused to a ligand-binding domain (LBD) through a hinge region. The LBD binds a small molecule ligand. Upon ligand binding, the LBD changes to an active conformation leading to the recruitment of coactivator (CoAC) proteins and initiation of transcription. As a result of their involvement in disease, there is an emphasis on engineering nuclear receptors for applications in gene therapy, drug discovery and metabolic engineering.

Hepatito B virusas (HBV) yra plačiai paplitęs žmogaus patogenas, bet iki šiol mažai ištirta jo šerdies baltymo (HBc), o ypač natūraliai aptinkamų mutantinių formų įtaka viruso dauginimuisi ir patogeniškumui. Šiame darbe mielių dviejų hibridų metodu atrinkti žmogaus kepenų baltymai, sąveikaujantys su laukinio tipo baltymu bei mutantais HBc1 ir HBc2. Su visomis tirtomis HBc atmainomis stipriausiai ir specifiškiausiai sąveikavo žmogaus baltymai GIPC1 ir GIPC2. Detaliau tiriant šias sąveikas nustatyta, kad HBc baltymo C-galas sąveikauja su GIPC1 ir GIPC2 baltymų PDZ domenais. HBc baltymo C-gale aptiktas PDZ domenų atpažįstamos sekos motyvas ir parodyta, kad šios sekos pokyčiai įtakoja HBc sąveiką su GIPC1 ir GIPC2. Vien su mutantais HBc1 ir HBc2 stipriausiai ir specifiškiausiai sąveikavo žmogaus baltymai FLJ20850 ir IKK (NEMO). Anksčiau netyrinėto nežinomos funkcijos žmogaus baltymo FLJ20850 raiška ir geno struktūra apibūdinta naudojantis bioinformatinėmis duomenų bazėmis. Detaliau tiriant mutantų sąveikas su FLJ20850 ir IKK buvo nustatytos baltymų sritys, apsprendžiančios tarpusavio sąveiką. IKK baltymas reguliuoja transkripcijos veiksnio NF-κB aktyvumą, todėl buvo tiriama ir mutanto HBc1 įtaka NF-κB aktyvumui žmogaus ląstelėse. Aptiktos baltymų sąveikos gali padėti geriau suprasti HBV dauginimosi ciklą bei patogeniškumą ir tapti naujų antivirusinių vaistų taikiniais. / Hepatitis B virus (HBV) is a major human pathogen, but up to now little is known about its core protein (HBc) interactions with host proteins. The role of mutated HBc proteins in enhanced pathogenicity of mutant viruses is also unclear. In this work, the yeast two-hybrid system was employed to find human proteins interacting with HBV core mutants HBc1 and HBc2, as well as with the wild-type core protein. All HBc variants strongly and specifically interacted with human proteins GIPC1 and GIPC2. Common protein interaction domain PDZ in both GIPC1 and GIPC2 was identified as the region interacting with the C-end of HBc. A putative PDZ-interacting motif was identified at the C-end of the HBc protein, and variation of this sequence influenced determined interactions. Human proteins FLJ20850 and IKKγ (NEMO) strongly and specifically interacted with mutants HBc1 and HBc2 only. Gene structure and expression FLJ20850 protein, which was never before described in scientific literature, were analyzed bioinformatically. Detailed analysis of interacting protein pairs revealed regions, responsible for discovered interactions. IKKγ is known as an important regulator of transcription factor NF-kB, therefore HBc1 influence on NF-kB activity in human cells was evaluated experimentally. Determined protein interactions potentially add to understanding of HBV replication and pathogenicity and could serve as targets for developing of new antivirals.

Hepatitis B virus (HBV) is a major human pathogen, but up to now little is known about its core protein (HBc) interactions with host proteins. The role of mutated HBc proteins in enhanced pathogenicity of mutant viruses is also unclear. In this work, the yeast two-hybrid system was employed to find human proteins interacting with HBV core mutants HBc1 and HBc2, as well as with the wild-type core protein. All HBc variants strongly and specifically interacted with human proteins GIPC1 and GIPC2. Common protein interaction domain PDZ in both GIPC1 and GIPC2 was identified as the region interacting with the C-end of HBc. A putative PDZ-interacting motif was identified at the C-end of the HBc protein, and variation of this sequence influenced determined interactions. Human proteins FLJ20850 and IKKγ (NEMO) strongly and specifically interacted with mutants HBc1 and HBc2 only. Gene structure and expression FLJ20850 protein, which was never before described in scientific literature, were analyzed bioinformatically. Detailed analysis of interacting protein pairs revealed regions, responsible for discovered interactions. IKKγ is known as an important regulator of transcription factor NF-kB, therefore HBc1 influence on NF-kB activity in human cells was evaluated experimentally. Determined protein interactions potentially add to understanding of HBV replication and pathogenicity and could serve as targets for developing of new antivirals. / Hepatito B virusas (HBV) yra plačiai paplitęs žmogaus patogenas, bet iki šiol mažai ištirta jo šerdies baltymo (HBc), o ypač natūraliai aptinkamų mutantinių formų įtaka viruso dauginimuisi ir patogeniškumui. Šiame darbe mielių dviejų hibridų metodu atrinkti žmogaus kepenų baltymai, sąveikaujantys su laukinio tipo baltymu bei mutantais HBc1 ir HBc2. Su visomis tirtomis HBc atmainomis stipriausiai ir specifiškiausiai sąveikavo žmogaus baltymai GIPC1 ir GIPC2. Detaliau tiriant šias sąveikas nustatyta, kad HBc baltymo C-galas sąveikauja su GIPC1 ir GIPC2 baltymų PDZ domenais. HBc baltymo C-gale aptiktas PDZ domenų atpažįstamos sekos motyvas ir parodyta, kad šios sekos pokyčiai įtakoja HBc sąveiką su GIPC1 ir GIPC2. Vien su mutantais HBc1 ir HBc2 stipriausiai ir specifiškiausiai sąveikavo žmogaus baltymai FLJ20850 ir IKK (NEMO). Anksčiau netyrinėto nežinomos funkcijos žmogaus baltymo FLJ20850 raiška ir geno struktūra apibūdinta naudojantis bioinformatinėmis duomenų bazėmis. Detaliau tiriant mutantų sąveikas su FLJ20850 ir IKK buvo nustatytos baltymų sritys, apsprendžiančios tarpusavio sąveiką. IKK baltymas reguliuoja transkripcijos veiksnio NF-κB aktyvumą, todėl buvo tiriama ir mutanto HBc1 įtaka NF-κB aktyvumui žmogaus ląstelėse. Aptiktos baltymų sąveikos gali padėti geriau suprasti HBV dauginimosi ciklą bei patogeniškumą ir tapti naujų antivirusinių vaistų taikiniais.